Target plate for mass spectometers and use thereof

ABSTRACT

A plate suitable for use with mass spectrometers comprising a number of target spots arranged in a surface portion of said plate making it possible to deposit small amounts of fluid at said target spots without the fluid escaping or getting mixed with fluid deposited at another target surface of the same plate. The target surfaces being arranged in a surface portion of said plate so that a base material of said plate constitutes the walls of receptacles, characterised in that the shape, size, temperature and possible agents of said receptacle facilitate evaporation of a solution in which sample molecules are suspended. Embodiments include different types of matrix and enzymes arranged at said spots, and methods for enhancing MALDI analysis efficiency.

FIELD OF INVENTION

The present invention relates to methods and devices for chemicalanalysis. More specifically it relates to methods and devices forpreparation of small amounts of sample molecules, facilitating asubsequent analysis using e.g. matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

BACKGROUND

The field of bioanalysis is experiencing an increased need for fast andaccurate devices/methods and also for devices/methods that are capableof establishing an accurate and precise analysis in spite of smallspecimen volumes.

Mass spectrometry involving ionisation by matrix-assisted laserdesorption (MALDI) has established itself as a standard procedure forthe analysis of biosubstances with large molecules. For this purpose,time-of-flight mass spectrometers (TOF-MS) are usually employed,although Fourier transform ion cyclotron resonance spectrometers(FT-ICR) or quadrupole ion trap mass spectrometers (in short: ion traps)are frequently/routinely used as ionisation principles for proteinsequencing.

In the following, the molecules of biosubstances to be studied will bereferred to simply as “analyte molecules” or “biomolecules”. In allcases, analyte molecules are present either in very diluted form inaqueous solutions, pure or mixed with organic solvents. Sometimes theseanalytical solutions are very complex and contaminated with respect tothe requirements of the analytical procedures, e.g., in the case of bodyfluids.

The biosubstances include all biopolymers and sometimes other substanceswith large molecules. “Biopolymers” comprise oligonucleotides (i.e.fragments of genetic material in various forms such as DNA or RNA),polysaccharides and proteins (the essential building blocks of theliving world) as well as their special analogues and conjugates such asglycoproteins or lipoproteins, and peptides arising from the action ofdigestive enzymes.

The selection of matrix substance for MALDI depends on the type ofanalyte molecule; more than a hundred different matrix substances arenow known. One of the tasks of the matrix substances include to isolatethe analyte molecules from each other wherever possible and bind them tothe sample carrier plate, to transfer the molecules into the vapourphase by forming a vapour cloud during the laser bombardment, andultimately to ionise the biomolecules by protonation or deprotonation,i.e., to add or remove one or more protons. For this task it has provenuseful to incorporate the analyte molecules individually in the crystalsof the matrix substances during their crystallisation, or at least tofinely distribute them in the boundary areas between the crystals. Hereit seems important to separate the analyte molecules from each other,i.e., no clusters of analyte molecules should be allowed in the preparedmatrix crystal sample.

A variety of procedures are known for applying analytes and matrices.The simplest of these entails the pipetting of a solution containingboth analyte and matrix onto a cleaned metallic sample support. The dropof solution wets a certain area of the metal surface (or its oxidelayer) whose size on hydrophilic surfaces is many times larger than thatof the diameter of a drop. The size depends on the hydrophilicity andthe microstructuring of the metal surface as well as on the propertiesof the droplet, in particular that of the solvent. After drying thesolution, a sample spot consisting of small matrix crystals and havingthe same size as that of the originally wetted surface area forms. Thematrix crystals are usually not uniformly distributed throughout theformerly wetted area. As a rule, crystals of the matrix start growing atthe inner margin of the wetting surface on the metal plate. They thengrow towards the interior of the wetting surface. They often form thinneedle crystals, as is the case for example for the frequently usedmatrices 5-dihydroxybenzoic acid (DHB) or 3-hydroxypicolinic acid (HPA),which often stand out from the carrier plate at the interior of thespot. The centre of the spot is frequently empty or covered with finecrystals, although they often cannot be used for MALDI ionisationbecause of their high concentration of alkaline salts. The loading ofthe crystals with biomolecules is also very uneven. This type of loadingtherefore requires viewing the sample carrier surface during MALDIionisation by a video microscope, which can be found in any commerciallyavailable mass spectrometer used for this type of analysis. Ion yieldand mass resolution vary in the sample spot from place to place. It isoften an arduous process to find a suitable position on the sample spotwith a satisfactory analyte ion yield and mass resolution, and onlyexperience, trial and error allow for improvements.

Although there are control programs for mass spectrometers withalgorithms for automatically seeking the best spots forMALDI-ionisation, such procedures, involving many attempts andevaluations, are necessarily very slow.

With other loading procedures the matrix substance is already present onthe carrier plate before application of the solvent droplets, which nowonly contain analyte molecules.

If the surface of the sample carrier plate is not hydrophilic, buthydrophobic, smaller crystal conglomerates are formed, and the dropletstend to wander in an uncontrollable manner during drying. Hence thelocalisation of the crystal conglomerates cannot be predicted and mustbe sought during the MALDI process. Furthermore, there is a considerablerisk that droplets will conglomerate and thus render a separate analysisof samples impossible.

Biosample analyses are now performed in their thousands, a situationwhich demands automatic high throughput procedures. A visual control orsearch, or even an automated search, would obstruct such a highthroughput procedure.

Recent prior art includes a procedure which leads to local andsize-defined crystallisation fields on small hydrophilic anchor regionsof 100 to 800 micrometer in diameter within an otherwise hydrophobicsurface (DE 197 54 978 C2). The aqueous drops are fixed by thehydrophilic anchors and prevented from wandering even when theyinitially rest on surrounding lyophobic areas. During drying thedroplets withdraw onto the anchor, and relatively dense, homogeneouslydistributed, crystalline conglomerates arise on the exact position ofthese anchors (sometimes even structured as a single compact crystallineblock depending on the type and concentration of matrix substance). Itcould be shown that the detection limit for analyte molecules improveswith reduction of the surface area of the wetting surface. Thus, smallerquantities of analytes and more diluted solutions can be worked withduring sample preparation; such an advantage is expressed in betterrunning biochemical preparatory procedures and reductions in chemicalmaterial costs. With a suitable preparation the analytical sensitivityover the surface of the sample is highly uniform. Thus the ionisationprocess can be freed from the need to perform visual or automatedsearches for favourable sites; instead a “blind” bombardment of thecrystal conglomerates with desorbing laser light can be used. Thispreparation method for prelocated spots of equal sensitivity acceleratesthe analytical process.

The crystal conglomerates forming on the hydrophilic anchor surfacesreveal a microcrystalline structure suitable for the MALDI-process. Asthe speed of the drying process is increased, the crystalline structurebecomes finer.

Here a “hydrophobic” surface is understood as a water repellant surface,i.e. one resistant to wetting by aqueous solutions. Correspondingly, a“hydrophilic” surface is understood as one that can be easily wetted bywater. “Oleophobic” and “oleophilic” (also referred to sometimes as“lipophobic” and “lipophilic”) refer to surfaces which repel or whichcan be wetted by oil, respectively. Organic solvents that are notmiscible with water usually have an oily nature in this meaning ofwettability, i.e. they can wet oleophilic faces. They are as a rulemiscible with oil. Organic solvents that are miscible with water, e.g.methanol, acetone or acetonitrile, can wet both oleophilic andhydrophilic surfaces in a pure state. However, the wettability ofoleophilic surfaces reduces as the water content increases.

For a long time it has been the opinion that hydrophobic surfaces arealways also oleophilic, and that oleophobic surfaces are alwayshydrophilic. However, for some years it has been known that surfacesexist which are both hydrophobic and oleophobic; these include smoothsurfaces of perfluorinated hydrocarbons such as polytetrafluoroethylene(PTFE). Such surfaces are designated here as “lyophobic”, a term whichhas been adopted from colloidal science.

Recently, it has also become known that the wetting or liquid repellingcharacter of a surface strongly depends on its microstructure. Anexample of this is the so called “lotus effect” (named after thelotus-plant).

A surface is particularly designated as “hydrophobic” when a dropretracts on a surface during drying or aspiration with a pipette,reducing the wetted surface reduces in size and leaving behind a drysurface (so called “dynamic hydrophobia”).

As a rule, biomolecules are best dissolved in water, sometimes with theaddition of organic, water-soluble solvents such as alcohol, acetone oracetonitrile. The analytical solutions of biomolecules sometimes alsocontain other substances such as glycols, glue-like buffering agents,salts, acids or bases depending on their preparation. The MALDI processis disrupted considerably by the presence of these impurities, sometimesthrough prevention of protonation, and sometimes through the formationof adducts. In particular, alkali ions often form adducts with analytemolecules of varying size and prevent any precise mass determination.The concentration of alkali ions in the sample preparation, as well asthe concentration of other impurity substances must be kept extremelylow by careful purification procedures.

For purification and simultaneous enrichment of biomolecules one can useso-called affinity adsorption media similar to those used in affinitychromatography. While in affinity chromatography one uses highlybioselective affinity adsorbents, for the purification of initiallyunknown mixtures of biopolymers without losses of special types ofbiomolecules, one needs non-specific adsorbents that can bind allbiomolecular constituents of the mixture to as near a similar degree aspossible.

For purification of peptides, proteins or DNA mixtures, sponge-likemicrospheres of adsorbent material (such as POROS, a registeredtrademark of Applied Biosystems, Inc.), pipette tips filled withsponge-like adsorbent (such as ZIPTIPs, a registered trademark ofMillipore Corporation) or C18 coated magnetised spheres (such asGenoPure, a product of Bruker Daltonics, Inc.) have proven particularlyuseful until now. These materials are all strongly oleophilic and bindpeptides or oligonucleotides via hydrophobic bonds. As a rule,biomolecules can be eluted using aqueous methanol or acetonitrilesolutions, and elution can often be assisted by altering the pH-value.However, purification with these materials is labour-intensive since itrequires additional materials and additional procedural steps.

Affinity capture methods have become known also for biospecificselection of certain biomolecules in connection with mass spectrometricanalysis, see e.g., U.S. Pat. Nos. 6,020,208, 6,027,942, or 5,894,063(T. W. Hutchens and T.-T. Yip). Such biospecific affinity adsorptionprocesses can be likewise used for purification.

In US application 20020045270A1 is disclosed a sample support plate withhydrophilic anchors in a strongly hydrophobic environment suitable forMALDI analysis. The plate provide areas with affinity adsorbentsadjacent to the hydrophilic anchors for purifying biosubstances and,optionally, for performing an affinity selection of biosubstances,whereby the finally prepared matrix sample crystals with thebiosubstances for the MALDI analysis are adequately localised on thehydrophilic anchors.

In a paper of Ekström et al., (Integrated micro-analytical technologyenabling rapid and automated protein identification (Anal. Chem. January2000)) is presented an integrated micro-analytical system, where amountsof samples are ejected onto a high-density nanovial MALDI-target plate.The so deposited sample is subsequently analysed by MALDI-TOF MS and theresulting peptide map is used for database search.

In a paper by Miliotis et al. (“Rapid Com. Mass Spect. 16, 2002, page117-126), is described matrix pre-coated nanovial MALDI targets, and ina paper by Ericsson (Ericsson D, Proteomics Vol 1, 2001, pages1072-1081) is described on-target nanovial digestion of proteins.

SUMMARY

Generally an object of the present invention is to provide a targetplate having spots for use in an array format and being provided withpre-positioned functions. Said plate also being devised to support atwo-dimensional read-out algorithm by protein sequencing and/or peptidemass fingerprinting. The target plate is preferably ready-made prior tosample deposition. This means that all necessary reagents and chemicalssuch as internal standards and crystallisation agents will be targetedon the plate prior to use. The two dimensional approach on the targetplate will be made in a way that e.g. 5 different crystallisation agentswill be used for the same sample. This will result in that differentsequences of the proteins will be detected by the 5 variouscrystallisation agents, thereby increasing the total sequence coverageof the proteins present in the samples.

Correspondingly and alternatively, embodiments of the invention cancomprise an additive dimension were e.g. 5 different enzymes will bedeposited. The different enzymes will have varying substrateselectivity. This will ultimately result in a differing cleavagespecificity whereby the resulting enzymatic product, the peptidecomposition will differ. Adding a second dimension of diversity in thearray target plate performance, these differing peptides from theenzymatic first dimension of analysis will then be analysed by the arrayof crystallisation agents. This will ultimately increase the versatilityof protein sequences and sequence coverage of the proteins analysed. Thepresent invention satisfies the initially mentioned needs. A targetplate and a method for use thereof and also a device for depositing anamount of sample on said plate is provided. The method together withsubsequent MALDI-TOF analysis and data base search is devised to givefast and accurate analysis results.

In a preferred embodiment a target plate having a target-plate surfaceis arranged to receive small, discrete and repeatable amounts of fluiddispensed from a micro dispensing device. Said target-plate surface isprovided with a two-dimensional array of target spots. Each spot isprovided with a spot agent, such that an amount of fluid received at aspot can interact with said agent. The agent can comprise a matrixsolution or alternatively a matrix solution together with one or moredigestive enzymes provided to enzymatically cleave analytes.

A dispensing control-unit is arranged to control the dispenser to shootat the right spot at a controlled pace and dispensing an appropriateamount of fluid for each spot. A temperature control unit is connectedto a target plate heater, said heater being provided for giving thetarget plate an appropriate temperature. When a burst of droplets isshot and subsequently is hitting a target spot, the heat makes the fluidto evaporate, leaving an increased-concentration of analyte moleculestogether with the agent enhancing the desired interaction between saidanalyte molecules and agent.

In another preferred embodiment a sample is divided into a number ofportions. Each portion is dispensed/shot to a separate spot. Each of theseparate spots is provided with a different agent, e.g. differentdigestive enzymes or different types of matrix. In the case withdifferent matrices, spots are arranged to receive different portions ofthe same sample. The spots are provided with different matrix solutionshaving different ionisation energy, such that (slightly) differentspectrograms is obtained for the same analyte, i.e., different portionsof the same sample, which enables increased specificity for theanalysis.

In another embodiment the agents on the spots can be different digestiveenzymes. A computer for matching the output from the mass spectrometeris provided with a database for identifying analysts in the sample. Saiddatabase is provided with spectrograms for a large number of knownsubstances/parts of substances that has been subjected to differentagents prior to mass spectrometry. Said computer is also arranged topresent the most plausible match, or matches (if any) to a person havinginterest in the result of the analysis.

Disposability

Embodiments of the present invention can easy provide disposable targetplates because they are expected to be cheap due to low costs ofmanufacturing by polymer materials. A further advantage of usingdisposable plates is that both the so-called carry over (contaminationstemming from earlier use, not totally removed during wash/clean) andthe so-called memory effect is eliminated.

DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where

FIG. 1 a,b,c and d shows a target plate with details of a nanovialincluding cross sections.

FIG. 2 shows a flowchart of a method for analysis using a target plate.

FIG. 3 shows a schematically a part of a MALDI-target plate withdeposited matrices and enzymes in a specific pattern.

DESCRIPTION

Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “annotate” is intended to mean the act of deciding that aspectrum of a MALDI spot corresponds to a certain biopolymer

The term “biopolymers” is intended to designate a group of substancescomprising, but not limited to, proteins, nucleic acids, andpolysaccharides.

The term “sample crystal” is intended to mean the dry result of chemicaland physical reactions concerning matrix and sample on the MALDI-Spot.

The term “MALDI Spot” is intended to mean an area at the MALDI targetplate for receiving and holding samples/sample crystals.

The term “matrix” is intended to mean a substance applied on theMALDI-spots prior to or at the same time or after the samples areapplied and which substance facilitates different aspects of theanalysis of the sample molecules, e.g., adherence to plate, distributionin space, absorption of laser energy.

The term “spectral data” is intended to mean data for a sample crystalcomprising the relative intensity and the mass/charge quotient for theions measured with a MALDI instrument during laser desorption/ionisationof said spot. Spectral data for a protein comprises the correspondingdata resulting from an identical MALDI analysis of a spot containingonly that protein in pure form. The present invention is directedtowards a target plate for preparing analyte samples for matrix-assistedlaser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). Alternative terms: spectrum, fingerprint.

The term “nanovial” is intended to mean small vials, in this contextpreferably on a MALDI-plate, and preferably comprising a MALDI-spot.Alternative terms: vial, well, spot

Target Plate

A preferred embodiment of the invention is shown in FIG. 1. A targetplate 100 is provided with a number of nanovials 110, 111, 112 etcregularly arranged at said plate. Each vial 110, 111 etc is providedwith walls 140, and a bottom surface, or spot 150. Alternativeembodiments include vials 160 of other shapes e.g. round, rounded, withslanting or elliptically shaped walls, and having a depth of no morethan 100 micrometers, preferably not more than 50 micrometers therebymatching the properties of MALDI laser beams in use today. Deeper vialscould affect MALDI-TOF mass resolution negatively.

Size and Arrangement of Spots

In an advantageous embodiment said walls are arranged to have a width of300 micrometer and a height of less than or equal to 50 micrometer,providing the vial with a maximum depth of 50 micrometer and giving thespot 150 an area of approximately 300 times 300 micrometer. This area isarranged to match with the effective cross-section area of laser beamsof commercially available MALDI lasers, such that the area of the spotis approximately within the range of 25 to 400 percent of laser beamcross-section area.

The spots are preferably arranged as a two-dimensional array with adistance between spots centre to centre so adapted as to coincide withcorresponding measures of commercially available high-density platesused in MALDI spectrometry. These centre to centre distances is alsopreferably such that centre to centre distances of dispenser nozzles ofan array dispenser or a dispenser array are made to correspond.

Plane Target Plate without Vials

An alternative preferred embodiment comprises a substantially planeplate having no vials or recesses of any kind, instead the plate isprovided with reference points which can comprise the corners and/oredges to facilitate proper application, processing and measurements ofthe molecules in correct areas of the plate. Analytes and/or othersubstances/fluids are kept in the right spot by means of surface tensionand/or surface modified surfaces such that two nearby amounts of fluidon two nearby spots do not mix even if the distance is 800 micrometerscentre to centre.

Method of Providing the Plate Spots with Agents

Commercially available methods for providing the plate spots with agentcan be used. In an alternative embodiment a micro dispenser is used todispense, i.e. shoot small droplets of agent towards the target plate,thereby providing it with the desired agent or pattern of agents

Use of Plate

Referring to FIG. 2, an embodiment of a method of using the target plate100 for analysis of samples comprises the following steps:

-   -   Loading 210 the target plate spots 150 with one or preferably        with a pattern of different MALDI-matrix solutions, i.e.        depositing a thin layer of matrix on each spot. The MALDI-matrix        solution is arranged to i) absorb energy and protect the analyte        from excessive energy during laser bombardment, i.e. to prevent        analyte decomposition. It is also arranged to ii) enhance ion        formation of the analyte by photoexcitation or photoionisation        of matrix molecules followed by proton transfer to the analyte        molecule and iii) said matrix is also arranged to dilute sample        into the matrix thereby preventing association of analyte        molecules.    -   (optional step) Loading 215 the target plate spots with none,        one or preferably with a pattern of different digestive enzymes.    -   Dispensing 220 analyte solutions on said loaded target plate        spots.    -   Facilitating 230 mixing of analyte and matrix or matrix/enzyme        solutions in each spot. (This will happen spontaneously at room        temperature when using droplet dispensing and increased mixing        speed is obtained in the arranged micro format.)    -   Allowing 240 said mixture to dry/solution to evaporate, forming        crystalline coating on each spot of the target plate.    -   Subjecting 250 the target plate to MALDI-TOF MS analysis        subsequently resulting in intensity versus m/z graphs, i.e. in        mass spectrograms, for each spot on the plate (m=mass, z=charge,        for ions of the analyte)    -   Matching 260 each spectrum against a database of known spectra.    -   Presenting 270 plausible matches

Preferably, the steps of dispensing 220, facilitating 230 and allowingto dry/solution to evaporate 240 is devised such that a first volume ofa protein containing substrate/solution, giving rise to a firstconcentration of proteins and, by the presence of enzymes, aconcentration of peptides, gives rise, because of the evaporation ofsolution/fluid, to a second concentration of proteins more favourablefor enzymatic cleavage than the first, cf. Michaelis-Menten equation ofenzyme kinetics.

The dispensing 220 is preferably performed using a micro dispenserhaving one or preferably a multitude of dispenser nozzles, making itpossible to dispense amounts of the same or different samples at thesame time, i.e., in parallel.

The action of the dispenser is controlled by a control unit thatsynchronises the action with the flow of analyte. The stepwise movementof the target plate for a next row of spots to be placed in front of(under) the dispenser nozzles is also synchronised with the actions ofthe dispenser.

The dispensing of droplets is conducted in symphony with the evaporationof the eluant so that the amount of analyte e.g. proteins, deposited onthe spot can be increased over time by dispensing more droplets on thesame spot. The evaporation is devised to take place in a temperature andin a so small volume that it becomes rapid, i.e. the most of thedeposited solution is evaporated within a few seconds.

In a preferred embodiment the spots are provided with enzymes that, dueto the small dimensions, the controlled temperature and the highconcentration of proteins, digest said proteins and form a highconcentration of peptides. A high concentration of peptide is favourablewhen performing a further chemical analysis by means of e.g. massspectrometry.

In this context it is possible to use the device to perform both globalexpression studies and focussed expression studies.

Activated Biofunctional Surfaces

In the case of activated biofunctional surfaces an additional washingstep 235 prior to MALDI analysis is performed.

Surface Modification of Target Plate Vials

In a preferred embodiment of the plate the vials/spots are modifiedusing one or more methods coming from the group comprising hydrophilicchemical modification, hydrophobic chemical modification, metalaffinitycoated chemical modification, antibody biochemical modification, antigenbiochemical modification, peptide biochemical modification, capturingbiomacromolecule modification.

Material

The device is preferably manufactured in silicon, glass or in a polymermaterial. Silicon is essentially inert when dealing with proteinmixtures at room or near room temperature. The material is also verysuitable for micro-machining techniques, e.g. for etching away parts ofthe material with established etching techniques.

Another advantage is that with said etching techniques the dimensionmeasures becomes very precise and it is possible to etch surface withfar better than micrometer precision.

The device can optionally be coated with gold or another highconductivity material in order to lead away charges. Alternatively oneor more conductive polymers can be used.

Method(s) for Improved Analysis Using Plate, MALDI Apparatus, andCorresponding spectral Databases and Search Algorithms

The spots with enriched analyte molecules arranged in an array format onthe target plate will be analysed in the MALDI mass spectrometer using asingle dimensional run (simple array format) which means that an arrayof chemical agents (matrices, enzymes) are screened.

Referring to FIG. 3, the sample positioning in the MALDI instrument willrun from position A01, A02 and so on. A real time data base search isperformed simultaneously with the ongoing analysis, where the identityof the protein is queried for by comparing the resulting spectrum, saidspectrum comprises the relative intensity and the mass/charge for theincorporated peptides. Automatic retrieval of the peptide sequencescomprising the proteins is accomplished by searching a database, alsocalled map, that has been compiled in advance.

Real-time identifications are also performed from position A01 where thespot is analysed for possible multi proteins present in the MALDI spot.Such real-time identifications are made by subtracting the peptidemasses/spectral data that belongs to the protein that was identified asa significant hit, and perform a second pass search where ongoingionisation in real-time is made by laser pulsing onto position A01 whereadditional peptides are ionised from the sample crystal spot. If asecond protein identity is confirmed a third pass search is made by theinstrument on the very same spot position, A01 subtracting the peptidemasses/spectra corresponding to both the first and second protein. Afterquerying the third pass in real-time, additional data base searches arenot performed by the instrument on the given target plate in theautomated run in this so-called real-time MALDI-target protein screeningcascade.

Moving from sample 1 to sample 2 on the target plate; Once theannotation can be confirmed as a high and statistically significantscore, the annotation of that protein sample is confirmed in real-timeand the instrument switches over to analyse position (spot) A02 on theMALDI sample plate.

Initially all samples are processed in the first dimension (“left toright”, A01, A02, A03 etc) screening through different chemical agents(matrices) in order to generate a multitude of protein crystallisationprocesses whereby the crystals will have varying physical-chemicalproperties that will give them different ionisation characteristicswhereby the specific peptides present in the sample will find theiroptimal ionisation and time-of-flight properties.

In case of low levels of protein sample present, the second dimension ofthe array (extended array format) will be initiated where differentenzymes have been used to digest the named protein sample. This meansthat sample position B01 will be analysed followed by B02 etc. that willhold the first chemical agent (B01), the second (B02) and so on. Thesame automated run screening through the target plate with thepre-positioned protein sample spots and performing real-time data basesearches on all the peptide masses generated by the MALDI instrumentwill subsequently be performed until statistically significantprotein(s) can be identified.

An automated feedback loop function in the processing will halt thescreening in the array once the fulfilment of protein sequence given hasbeen obtained. The requirement for protein sequence will be determinedby the operator of the study.

The array dispenser will be operated in a number of functions togetherwith the target plate;

-   -   Static mode Sample array mapping    -   Separation mode of array mapping

In the Static mode a single sample will be fed into all the nozzles ofthe array, thereby dispensing a subset of the same sample onto both thefirst (chemical agent matrix), and the second dimension differentenzymes of the target plate array.

In the Separation mode, new proteins will be deposited onto the targetplate in the two dimensions with time.

Dispensing sample droplets can be accomplished with a dispenser array orwith an array of dispensers such that simultaneous array depositionprovides reduced experimental variation in-between sample spots.

1. A plate suitable for use with mass spectrometers comprising a numberof vials and/or target spots arranged in a surface portion of said platemaking it possible to deposit small amounts of fluid at said targetspots without the fluid escaping or getting mixed with fluid depositedat another target surface of the same plate wherein one or more agentsare arranged on said each target spot, said one or more agents are amatrix, an enzyme or a highly selective biomacromolecule affinity binderor a mixture thereof, in such a way that different target spotscomprises different agent mixtures, said mixtures comprising differentmatrices and/or different enzymes with varying substrate selectivity,resulting in a differing cleavage specificity such that a subsequentanalysis with MALDI of the enzymatic product, can make use of theinformation arising from the resulting slightly different spectra.
 2. Aplate as recited in claim 1, wherein said agents comprise digestionenzymes, such that, because of small dimensions, controlled temperatureand sufficiently high concentration of applied protein samples after theequilibrium of the enzymatic reaction the resulting sample will containa high concentration of peptides.
 3. A plate as recited in claim 2, saidtarget surfaces being arranged in a surface portion of said plate sothat a base material of said plate constitutes the walls of receptacles,wherein the shape, size and temperature of said receptacle facilitateevaporation of a solution in which sample molecules are suspended.
 4. Aplate as recited in claim 3, wherein a diameter of the spot isapproximately between 300 to 400 micrometers such that it matches thediameter of MALDI laser beams in a relationship of approximately 3 to 1,ranging to 4 to
 1. 5. A plate as recited in claim 4, where said shapeand size conform to the following limitations: when viewed from abovesaid receptacle is rectangular in shape; the spot comprises the shape ofa rectangular parallelepiped.
 6. A plate as recited in claim 5, wheresaid shape of receptacle comprises a rounded cross section profile.
 7. Aplate as recited in claim 6, wherein said plate comprises target meansmaking it especially suitable for receiving sample droplets dispensedfrom a dispenser.
 8. A plate as recited in claim 7, wherein saiddispenser is a dispenser array or an array dispenser, such thatsimultaneous array deposition provides reduced experimental variation inbetween sample spots.
 9. A plate as recited in claim 8, wherein saidplate comprises target spots arranged in columns and rows and in thatthe target spots in a column each is provided with the same matrix, andthat the target spots in a different column are provided with adifferent matrix.
 10. A plate as recited in claim 9, wherein said platecomprises target spots arranged in columns and rows and in that thetarget spots in a row each is provided with the same enzyme, and in thatthe target spots in a different row are provided with a differentenzyme, such that, in general, no target spot is provided with the samematrix-enzyme mixture.
 11. A plate as recited in claim 10, wherein saidplate is devised to be disposable.
 12. A method for preparing a platefor subsequent MALDI-analysis, comprising the steps of: arranging, bydispensing, matrices in target spots, such that the target spots in acolumn each is provided with the same matrix, and that the target spotsin a different column are provided with a different matrix, applicationof a first volume of a protein containing substrate/solution on one ofsaid spots, evaporation of said solution and enzymatic cleavage, suchthat said application gives rise to a first concentration of proteinsand, by the presence of enzymes, a concentration of peptides, givingrise to, because of the rapid evaporation of solution/fluid, a secondconcentration of proteins more favourable for enzymatic cleavage thanthe first.
 14. The method according to claim 12 further comprising thestep of arranging, by dispensing, enzymes in target spots such that thetarget spots in a row each is provided with the same enzyme, and in thatthe target spots in a different row are provided with a different enzymesuch that, in general, no target spot is provided with the samematrix-enzyme mixture.
 15. The method according to claim 13 for applyinga mixture of agent and analyse on a target plate spot comprising thatthe analyte is applied first.
 15. The method according to claim 14 forapplying a mixture of agent and analyte on a target plate spotcomprising that the agent is applied first.
 16. The method according toclaim 15 for applying a mixture of agent and analyte on a target platespot comprising that the agent and analyte is applied simultaneously.